The present invention relates to improved lentivirus-derived packaging and transducing vectors useful for the expression of genes at high levels in eukaryotic cells. The improved vectors are safer, yet permit increased efficiency of packaging the recombinant viral genome and increased long-term gene expression.
Viral vectors transduce genes into target cells with high efficiencies owing to specific virus envelope-host cell receptor interaction and viral mechanisms for gene expression. Consequently, viral vectors have been used as vehicles for the transfer of genes into many different cell types including whole embryos, fertilized eggs, isolated tissue samples, and cultured cell lines. The ability to introduce and express a foreign gene in a cell is useful for the study of gene expression and the elucidation of cell lineages (J. D. Watson et al., Recombinant DNA, 2d Ed., W. H Freeman and Co., NY [1992], pp. 256-263). Retroviral vectors, capable of integration into the cellular chromosome, have also been used for the identification of developmentally important genes via insertional mutagenesis (J. D. Watson et al., supra, p. 261). Viral vectors, and retroviral vectors in particular, are also used in therapeutic applications (e.g., gene therapy), in which a gene (or genes) is added to a cell to replace a missing or defective gene or to inactivate a pathogen such as a virus.
In view of the wide variety of potential genes available for therapy, it is clear that an efficient means of delivering these genes is sorely needed in order to fulfill the promise of gene therapy as a means of treating infectious, as well as non-infectious diseases. Several viral systems including murine retrovirus, adenovirus, parvovirus (adeno-associated virus), vaccinia virus, and herpes virus have been developed as therapeutic gene transfer vectors (For review see, A. W. Nienhuis et al., Hematology, Vol. 16:Viruses and Bone Marrow, N. S. Young (ed.), pp. 353-414 [1993]).
Factors affecting viral vector usage include tissue tropism, stability of virus preparations, genome packaging capacity, and construct-dependent vector stability. In addition, in vivo application of viral vectors is often limited by host immune responses against viral structural proteins and/or transduced gene products.
One of the key issues in human gene therapy is the toxicity and safety to the treatment subjects. Gene therapy applications in humans have met with problems associated with the host immune responses against the gene delivery vehicles or the therapeutic gene products. Viral vectors (e.g., adenovirus) which co-transduce several viral genes together with the therapeutic gene(s) are particularly problematic. For example, readministration is necessary for adenovirus vectors because of the transient nature of viral gene expression. As such, a host immune response to the vector or the therapeutic gene product may be detrimental (B. C. Trapnell and M. Gorziglia, Curr. Op. Biotechnol., 5:617-625 [1994]; and S. K. Tripathy et al., Nature Med., 2:545-550 [1996]).
Although MLV vectors have not been reported to induce cytotoxicity and do not elicit strong host immune responses, lentiviral vectors such as HIV-1 which carry several immunostimulatory gene products have the potential to cause cytotoxicity and induce strong immune responses in vivo. The latter are known to induce strong cell-mediated immune responses upon transient exposure (M. Clerici et al., J. Inf. Dis., 165:1012-1019 [1992]; M. Clerici et al., J. Amer. Med. Assoc., 271:42-46 [1994]; L. A. Pinto et al., J. Clin. Invest., 96:867-876 [1995]; and S. Rowland-Jones et al., Nature Med., 1:59-64 [1995]). However, this may not be a concern for lentiviral derived transducing vectors, as the latter need not encode any viral genes in the transducing vector.
Of course, in some instances, the purpose of the vector is to provoke a clinically useful immune response against an encoded protein.
Another important issue related to the lentiviral vector usage is that of possible cytopathogenicity upon exposure to some cytotoxic viral proteins. Exposure to HIV-1 proteins may induce cell death or functional unresponsiveness in T cells (N. Chirmule et al., J. Virol., 69:492-498 [1995]; C. J. Li et al., Science 268:429-431 [1995]; J. D. Lifson et al., Science 232:1123-1127 [1986]; I. G. Macreadie et al., Mol. Microbiol., 19:1185-1192 [1996]; and T. Nosaka et al., Exp. Cell. Res., 209:89-102 [1993]). During the development of the present invention, it was observed that direct gene transfer into tissue culture cells by the calcium-phosphate DNA co-precipitation method could induce more than 80% cell death which is caused mainly by necrosis and a residual percentage, approximately 2-4%, by programmed cell death
A final concern is the possibility of generating replication-competent, virulent virus by recombination.
Safety concerns have prompted much effort towards the development of non-viral vector systems, such as liposome-mediated gene transfer, naked DNA injections and gene gun technology. However, all of these non-viral gene transfer methods lack the ability to allow permanent integration of foreign genes into the host cell chromosomes, and are relatively inefficient. For long term expression of therapeutic genes in target cells, efficient means of transduction and genome integration are essential.
The term xe2x80x9cretrovirusxe2x80x9d is used in reference to RNA viruses that utilize reverse transcriptase during their replication cycle. The retroviral genomic RNA is converted into double-stranded DNA by reverse transcriptase. This double-stranded DNA form of the virus is capable of being integrated into the chromosome of the infected cell; once integrated, it is referred to as a xe2x80x9cprovirus.xe2x80x9d The provirus serves as a template for RNA polymerase II and directs the expression of RNA molecules which encode the structural proteins and enzymes needed to produce new viral particles. At each end of the provirus are structures called xe2x80x9clong terminal repeatsxe2x80x9d or xe2x80x9cLTRs.xe2x80x9d The LTR contains numerous regulatory signals including transcriptional control elements, polyadenylation signals and sequences needed for replication and integration of the viral genome. There are several genera included within the family Retroviridae, including Cisternavirus A, Oncovirus A, Oncovirus B, Oncovirus C, Oncovirus D, Lentivirus, and Spumavirus. Some of the retroviruses are oncogenic (i.e., tumorigenic), while others are not. The oncoviruses induce sarcomas, leukemias, lymphomas, and mammary carcinomas in susceptible species. Retroviruses infect a wide variety of species, and may be transmitted both horizontally and vertically. They are integrated into the host DNA, and are capable of transmitting sequences of host DNA from cell to cell. This has led to the development of retroviruses as vectors for various purposes including gene therapy.
Retroviral vectors derived from the amphotropic Moloney murine leukemia virus (MLV-A), use cell surface phosphate transporter receptors for entry and then permanently integrate into proliferating cell chromosomes. The amphotropic MLV vector system has been well established and is a popular tool for gene delivery (See e.g., E. M. Gordon and W. F. Anderson, Curr. Op. Biotechnol., 5:611-616 [1994]; and A. D. Miller et al., Meth. Enzymol., 217:581-599 [1993]).
Other retroviruses, including human foamy virus (HFV) and human immunodeficiency virus (HIV) have gained much recent attention, as their target cells are not limited to dividing cells and their restricted host cell tropism can be readily expanded via pseudotyping with vesicular stomatitis virus G (VSV-G) envelope glycoproteins (See e.g., J. C. Burns et al., Proc. Natl. Acad. Sci. USA 90:8033-8037 [1993]; A. M. L. Lever, Gene Therapy. 3:470-471 [1996]; and D. Russell and A. D. Miller, J. Virol., 70:217-222 [1996]). However, a useful lentiviral vector system has not been well established, mainly because of the lack of sufficient studies on lentiviral vectorology and safety concerns.
While many viral vector systems are available, virtually all of the current human gene therapy trials use retroviral vectors derived from the amphotropic Moloney murine leukemia virus (M-MuLV), such as pLNL6 (Genbank M63653), see Baker, et al., J. Virol. 61:1639 (1987), for gene transfer (see also A. D. Miller and C. Buttimore, Mol. Cell. Biol., 6:2895 [1986]). Among the vectors known in the art, special note may be taken of Chang, U.S. Pat. No. 5,693,508 (1997) which discloses retroviral vectors contining chimeric MoMLV/CMV-IE/HIV-TAR LTRs. The elements essential to the retroviral vector system are viral structural proteins Gag, Pol and Env, the long terminal repeats (LTR), the reverse transcription templates including primer binding site (PBS) and polypurine tract (PPT), and the packaging signals (psi ["psgr"]). The MLV-A vector system is comprised of a packaging cell line expressing Gag, Pol and Env, and a vector construct containing LTRs, PBS, PPT and the packaging signal sequences. Up to 8 kbp of foreign sequences can be inserted into the MLV vector and packaged into virus particles. The commonly used amphotropic MLV packaging cell lines such as PA317, PG-13, "psgr"-CRIP, GP-AM12 and FLY-A13 produce 105-107 transducing units per ml after vector DNA transfection (F.-L. Cosset et al., J. Virol., 69:7430-7436 [1995]; H. Kotani et al., Human Gene Ther., 5:19-28 [1994]; J. S. Lam et al., Human Gene Ther., 7:1415-1422 [1996]; D. Markowitz et al., J. Virol., 62:1120-1124 [1988]; A. D. Miller and F. Chen, J. Virol., 70:5564-5571 [1996]).
The M-MuLV system has several advantages: 1) this specific retrovirus can infect many different cell types; 2) established packaging cell lines are available for the production of recombinant M-MuLV viral particles; and 3) the transferred genes are permanently integrated into the target cell chromosome. The established M-MuLV vector systems comprise a DNA vector containing a small portion of the retroviral sequence (the viral long terminal repeat or xe2x80x9cLTRxe2x80x9d and the packaging or xe2x80x9cpsixe2x80x9d ["psgr"] signal) and a packaging cell line. The gene to be transferred is inserted into the DNA vector. The viral sequences present on the DNA vector provide the signals necessary for the insertion or packaging of the vector RNA into the viral particle and for the expression of the inserted gene. The packaging cell line provides the viral proteins required for particle assembly (D. Markowitz et al., J. Virol., 62:1120 [1988]).
The vector DNA is introduced into the packaging cell by any of a variety of techniques (e.g., calcium phosphate coprecipitation, lipofection, electroporation, etc.). The viral proteins produced by the packaging cell mediate the insertion of the vector sequences in the form of RNA into viral particles which are shed into the culture supernatant. The M-MuLV system has been designed to prevent the production of replication-competent virus as a safety measure. The recombinant viral particles produced in these systems can infect and integrate into the target cell but cannot spread to other cells. These safeguards are necessary to prevent the spread of the recombinant virus from the treated patient and to avoid the possibility of helper virus-induced disease (A. D. Miller and C. Buttimore, supra; and D. Markowitz et al., supra).
After selection, producer cell clones can be established to generate 104-106 transducing units per ml. Increased transduction efficiencies may be achieved by modification of the transduction protocols through means such as repetitive infection steps, cocultivation with the producer cell line, centrifugation, and modification of the culture conditions using growth factors and fibronectin etc. (H. Kotani et al., Human Gene Ther., 5:19-28 [1994]; and T. Moritz et al., Blood 88:855-862 [1996]).
Despite these advantages, existing M-MuLV-based retroviral vectors are limited by several intrinsic problems: 1) they do not infect non-dividing cells (D. G. Miller et al., Mol. Cell. Biol., 10:4239 [1990]); 2) they produce only low titers of the recombinant virus (A. D. Miller and G. J. Rosman, BioTechn., 7: 980 [1989]; and A. D. Miller, Nature 357: 455 [1992]); 3) they express foreign proteins at low levels and often get xe2x80x9cturned-offxe2x80x9d or inactivated after integration (A. D. Miller, Nature 357: 455 [1992]); (4) the instability of the enveloped virus particles, as it is both difficult to concentrate in vitro and difficult to manipulate in vivo (A. D. Miller, Nature 357:455-460 [1992]); 5) the MLV LTR activity is also known to be suppressed in embryonal cells (P. M. Challita et al., J. Virol., 69:748-755 [1995]; and T. P. Loh et al., J. Virol., 62:4086-4095 [1988]); and 6) long term expression after viral integration is often restricted by transcription repression, likely due to DNA methylation (J. Boyes and A. Bird, Cell 64:1123-1134 [1991]; and M. Szyf et al., Mol. Cell. Biol., 10:4396-4400 [1990]).
The low production of recombinant virus produced by the M-MuLV system (e.g., 106/ml) compared to the adenoviral system (up to 1012/ml) means that human cells are infected at a very low efficiency. This low efficiency is particularly problematic when the target cell type is represented at very low numbers in the tissue to be infected. Although the hematopoietic stem cell is a preferred target for gene therapy in a large number of disorders, these cells are present at very low frequencies. For example, totipotent embryonic stem cells have been reported to occur at a frequency of 10xe2x88x924 to 10xe2x88x926 in bone marrow (B. R. Glick and J. J. Pasternak, Molecular Biotechnology, American Society for Microbiology, Washington, D.C., p. 412 [1994]). Thus, the low titer produced by existing M-MuLV vector systems is highly problematic for stem cell infection.
The promoter present in the M-MuLV LTR is quite weak compared with other viral promoters such as the human cytomegalovirus immediate early (CMV-IE) enhancer/promoter. In order to increase expression of the genes carried on the retroviral vector, internal promoters possessing stronger activities than the M-MuLV promoter have been utilized. However, the inclusion of an internal promoter to drive the expression of the inserted gene does not always lead to increased levels of expression (D. Robinson et al., Gene Therapy 2:269 [19951). Apparently, the activity of the internal promoter is significantly decreased because of interference from the upstream M-MuLV promoter (i.e., transcriptional read-through interference). The dual transcription-unit construct is, however, a common feature in almost all M-MuLV vectors.
To create an improved retroviral vector suitable for a wide variety of gene expression studies and gene therapy applications, the clinically approved gene therapy vector pLNL6 has been modified to allow synthesis of high basal levels of mRNA, and increased packaging efficiency (See e.g., co-pending U.S. patent application. Ser. No. 08/336,132, now U.S. Pat. No. 5,693,508, and PCT/US95/14576, to Chang, herein incorporated by reference). However, other limitations remain.
Given these limitations, it is clear that improved vector systems are urgently needed to provide a means of delivering and expressing genes efficiently in mammalian cells, particularly human cells. Improved vectors will aid the study of gene expression and development and are necessary if the promise of gene therapy is to be realized.
The major limitation in the use of the simple retroviral vectors in gene transfer is that use of the MLV-based vector is restricted to dividing cells. This led to the development of the present invention, in which lentiviruses, capable of infecting non-dividing cells are provided.
As used herein, the term xe2x80x9clentivirusxe2x80x9d refers to a group (or genus) of retroviruses that give rise to slowly developing disease. Viruses included within this group include HIV (human immunodeficiency virus; including HIV type 1, and HIV type 2), the etiologic agent of the human acquired immunodeficiency syndrome (AIDS); visna-maedi, which causes encephalitis (visna) or pneumonia (maedi) in sheep, the caprine arthritis-encephalitis virus, which causes immune deficiency, arthritis, and encephalopathy in goats; equine infectious anemia virus, which causes autoimmune hemolytic anemia, and encephalopathy in horses; feline immunodeficiency virus (FIV), which causes immune deficiency in cats; bovine immune deficiency virus (BIV), which causes lymphadenopathy, lymphocytosis, and possibly central nervous system infection in cattle; and simian immunodeficiency virus (SIV), which cause immune deficiency and encephalopathy in sub-human primates. Diseases caused by these viruses are characterized by a long incubation period and protracted course. Usually, the viruses latently infect monocytes and macrophages, from which they spread to other cells. HIV, FIV, and SIV also readily infect T lymphocytes (i.e., T-cells).
Lentivirus virions have bar-shaped nucleoids and contain genomes that are larger than other retroviruses. Lentiviruses use tRNAlys as primer for negative-strand synthesis, rather than the tRNApro commonly used by other infectious mammalian retroviruses. The lentiviral genomes exhibit homology with each other, but not with other retroviruses (See, Davis et al., Microbiology, 4th ed., J. B. Lippincott Co., Philadelphia, Pa. [1990], pp. 1123-1151). An important factor in the disease caused by these viruses is the high mutability of the viral genome, which results in the production of mutants capable of evading the host immune response. It is also significant that they are capable of infecting non-dividing cells.
Lentiviruses including HIV, SIV, feline immunodeficiency virus (FIV) and equine infectious anemia virus (EIAV) depend on several viral regulatory genes in addition to the simple structural gag-pol-env genes for efficient intracellular replication. Thus, lentiviruses use more complex strategies than classical retroviruses for gene regulation and viral replication, with the packaging signals apparently spreading across the entire viral genome. These additional genes display a web of regulatory functions during the lentiviral life cycle. For example, upon HIV-1 infection, transcription is up-regulated by the expression of Tat through interaction with an RNA target (TAR) in the LTR. Expression of the full-length and spliced mRNAs is then regulated by the function of Rev which interacts with RNA elements present in the gag region and in the env region (RRE) (S. Schwartz et al., J. Virol., 66:150-159 [1992]). Nuclear export of gag-pol and env mRNAs is dependent on the Rev function. In addition to these two essential regulatory genes, a list of accessory genes, including vif, vpr, vpx, vpu, and nef, are also present in the viral genome and their effects on efficient virus production and infectivity have been demonstrated, although they are not absolutely required for virus replication (K. and F. Wong-Staal, Microbiol. Rev., 55:193-205 (1991]; R. A. Subbramanian and E. A. Cohen, J. Virol. 68:6831-6835 [1994]; and D. Trono, Cell 82:189-192 [1995]).
HIV-1 virions contain 60% protein and 2% nucleic acid. The genome consists of two molecules of linear positive-sense single stranded RNA (held together by hydrogen bonds to form a dimer). Even within a single virion, these molecules need not be identical. Hence, genetic variation can occur through recombination between the two viral RNAs of a single virion.
The HIV-1 genome is about 9.7 kb in length. Many HIV-1 proviral genome sequences have been sequenced in their entirety. The sequence GenBank M19921, LOCUS HIVNL43, Human immunodeficiency virus type 1, NY5/BRU (LAV-1) recombinant clone pNL4-3, 9709 bp ss-RNA, is used as a reference sequence in this discussion. The construction of pNL4-3 has been described in Adachi, A., Gendelman, H. E., Koenig, S., Folks, T., Willey, R., Rabson, A. and Martin, M. A., Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone, J. Virol. 59, 284-291 (1986). pNL4-3 is a recombinant (infectious) proviral clone that contains DNA from HIV isolates NY5 (5xe2x80x2 half) and BRU (3xe2x80x2 half). The site of recombination is the EcoRI site at positions 5743-5748. The final sequence is set forth in Dai, L. C., Littaua, R., Takahashi, K. and Ennis, F. A., Mutation of human immunodeficiency virus type 1 at amino acid 585 on gp41 resultis in loss of killing by CD8+ A24-restricted cytotoxic T lymphocytes, J. Virol. 66, 3151-3154 (1992).
For several reasons, the HIV-1 genome has a high mutation rate. First, there can be recombination between the two RNAs of a single virion. Secondly, a single cell can be infected by more than one viral particle simultaneously, and recombination occur between the two viral genomes. Finally, the HIV reverse transcriptase has a high frequency of misincorporation (:1700 to 1:4000). The replication error rate for HIV is such that each newly synthesized HIV genome carries on average approximately one mutation. For all of these reasons, there is not one HIV-1 sequence, but rather a family of closely related sequences. Different HIV-1 sequences may be identified even in different samples isolated from a single individual. The degree of genetic variation observed is phenomenalxe2x80x94up to 20% within an infected individual. This is essentially due to remorseless cycles of viral replication, most probably due tochronic activation of the immune system. It can be estimated that the number of variants in existence worldwide must be in excess of 10(14)-10(18), and given the nature of RNA viruses even more novel variants should emerge.
HIV-1""s are currently divided into two genetic groups based on phylogenetic reconstruction using DNA sequences. The majority of these sequences fall into the M (major) group, while a smaller, but growing, number of sequences are classified as O (outlier). Most HIV-1 strains from around the world can be placed into one of nine nucleotide sequence-defined clades; these clades have been given the letter designations A through I. However, more than a dozen HIV-1 strains isolated from patients have now been shown to have chimeric genomes in that their gag and env genomic regions cluster with different clades. Interclade recombination is relatively easy to demonstrate because strains from different clades typically differ substantially in their nucleotide sequence identities. For example, the env gene sequences of HIV-1 strains of different clades may differ by 20% or more. As might be expected, interclade HIV-1 recombinants have most often been detected in geographic regions where two or more clades are prevalent. At least 17 HIV clades have now been reported in humans: nine HIV-1 clades in the major grouping (A through I), three HIV-1 group O group xe2x80x9coutlierxe2x80x9d clades, and five HIV-2 clades. An additional three lentiviruses are known in nonhuman primate species (African green monkeys, mandrils, and Syke""s monkeys). Thus the potential gene pool for primate lentivirus recombination is on the order of 20, e.g., 20 gag genes and 20 pol genes. The current HIV-1 clades may have arisen in part through past recombination between some of these genes. Viable recombinants between SIV and HIV (xe2x80x9cSHIVxe2x80x9d strains) have been genetically engineered in research laboratories.
The principal elements of the HIV-1 genome are set forth below, in the 5xe2x80x2 to 3xe2x80x2 direction. For further information, see Vaishnav and Wong-Staal, Ann. Rev. Biochem., 60: 577-630 (1991). The positions of each element are given according to the Genbank numbering of the complete genome sequence (M19921) cited above. That means that the numbering begins with the first base of the 5xe2x80x2 LTR, not with the cap site. The exact positions will vary from strain to strain, and some elements are better defined than others. Note that some genetic elements overlap, and that two (Tat and Rev) are interrupted. For a compilation of numerous sequences and alignments, at both the nucleic acid and amino acid levels, for many lentiviruses and othe retroviruses, see the HIV Sequence Database at http://hiv-web.lanl.gov.
5xe2x80x2 LTR (1-634)
Each end of the DNA provirus contains the so-called long terminal repeats (LTRs). The 5xe2x80x2 LTR and 3xe2x80x2 LTR regions are essentially identical in the wild-type HIV-1 genome. These LTRs are 634-bp non-coding sequences, located at the extreme 5xe2x80x2 and 3xe2x80x2 ends of the proviral genome, that contain enhancer and promoter regions. The LTRs consist of three distinct coding regions, U3, R, and U5, which can be subdivided into the separate enhancer and promoter regions. The U3 region is 450, the R sequence 100 and the U5 region some 85 nt long. Transcription initiates at the first base of the R region in the 5xe2x80x2 LTR, and polyadenylation occurs immediately after the last R region base in the 3xe2x80x2 LTR. The primary transcript is thus about 600 bases shorter than the provirus.
The U3 region includes several features of interest: the integration attachment site (att) at the far 5xe2x80x2 end, the promoter TATA box (a segment of DNA, located approximately 19-27 base pairs upstream from the start point of eukaryotic structural genes, to which RNA polymerase binds), promoter (SP1) regions (promoter binding site for RNA polymerase and reverse transcriptase), the kappa-enhancer (contains two imperfect 11-bp repeats, GGGACTTTC(SEQ ID NO.58), and IL-1 and IL-2 homologous enhancers.
The R region (454-550) contains the transcription initiation site, the TAR (Tat-activating) region and the poly A signal (xe2x80x94AATAAAAxe2x80x94); the latter is significant only in the 3xe2x80x2 LTR). The primary transcript corresponds to bases 455 to 9626.
The U5 region contains a polyA downstream element and a second integration attachment site at the 3xe2x80x2 end. These are significant only in the 3xe2x80x2 LTR. PBS
Immediately downstream of the 5xe2x80x2 LTR is the primer binding site (637-651) for minus-strand DNA synthesis, called the RNA cap. The primer binding site is complementary to the 3xe2x80x2 end of a Lys transfer RNA.
5xe2x80x2 Leader (L)
The 5xe2x80x2 leader (L), the untranslated region between the primer binding site and the initiation codon for gag, has two elements worthy of note.
The first is the major 5xe2x80x2 splice donor (SD) site (the splice point is at 748) which is used for the processing of full-length genomic RNA to subgenomic mRNA for the syntheses of various viral proteins. The major splice donor site is so called because it acts as the donor site during splicing of the vif, vpr, tat, rev, vpu-env and nef subgenomic RNAs (The Gag-Pol polyprotein is translated from genomic RNA.) There are also minor splice donor sites in the vicinity of the first exon of the rev gene.
The other is the major packaging signal (psi) (651-669) which serves as a contact point for the Gag nucleocapsid (Ncp7) protein to bind the RNA and to incorporate it into virus particles. Note that one can define an extended packaging signal extending into the gag gene, to about 820.
The 5xe2x80x2 leader also contains a sequence which participates in the dimer-linkage structure of 70S RNA. This DLS overlaps with the major packaging signal.
A secondary structure model of the leader, and the 5xe2x80x2 end of gag, was prepared by Baudin, et al., J. Mol. Biol., 229: 382-97 (1993).
Structural Genes
The gag gene encodes a polyprotein (55 kDa) (CDS 790..2292) which is cleaved by the viral protease (see pol) to yield various core and nucelocapsid proteins. The gag coding region extends from the ATG initiation codon at nucleotide 337 to nucleotide 1837 relative to the RNA cap site. The polyprotein is translated from unspliced viral RNA. The precursor Gag protein is cleaved by protease to produce p17 (the major matrix MA protein, involved in membrane anchoring, env interaction, and nuclear transport of viral core), p24 (the core capsid CA protein), p7 (the nucleocapsid NC protein, which binds RNA), and p6 (which binds Vpr). A pair of zinc finger motifs in the NC protein binds to the major packaging signal in the viral RNA.
The gag gene is believed by some authors to contain one or more minor packaging signals.
The pol gene (CDS est. 2085..5096) codes for a large polyprotein which is a precursor to the virion proteins providing the viral enzyme functions: protease, reverse transcriptase, and integrase. The gag and pol genes overlap by 241 nucleotides, and are in different reading frames. A slippage sequence in or upstream of the gag-pol overlap region induces an occasional ribosomal frameshift at a frequency (about 5%) which ensures that Gag proteins are made in large amounts and Pol proteins in small amounts. Initially, a gag-pol fusion protein (p190) is created as a result of the ribosomal frameshift, which does not interrupt translation. The viral protease cleaves Gag from Pol, and further digests Gag and Pol to separate the various mature proteins. In the case of Pol, the cleavage products are protease (p10), reverse transcriptase (p50), Rnase H (p15) and integrase (p31). Roughly 50% of the RT remains linked to Rnase H as a single polypeptide (p66). The principal functional form of RT is actually a heterodimer of p66 and p50. All pol gene products are found within the capsid of free HIV-1 virions.
Reverse transcriptase is responsible for the synthesis of double-stranded DNA from the viral RNA. Activity of RT is localized to the N-terminus. RT in HIV has an extremely high error rate, 1/1700 nucleotides. At the 3xe2x80x2 end of the pol coding region is the coding region for viral endonuclease/integrase. Integrase functions to integrate the proviral DNA in the host genome.
The env gene (CDS 6221..8785) is located at the 3xe2x80x2 end of the genome. It encodes the envelope protein gp160, some of which is cleaved to yield the envelope proteins gp120 and gp41. Both function in cell recognition on the outer envelope of a released virus. The C-terminus of gp120 interacts with the viral receptor CD4 of human T lymphocytes to facilitate the viral entry into the host cell. Only a 12 amino acid sequence in gp120 is necessary for binding to CD4; the rest of the protein is mutable. The gp120 polypeptide contains nine conserved intrachain disulfide bridges and, within this scaffolding, folds into five globular domains (I-V). There are five hypervariable regions (V1-V5) whose sequences vary especially widely among HIV-1 isolates.
Regulatory Genes
The tat gene (CDS 5830..6044, 8369..8414) encodes Tat, a trans-activating protein, the most important activator of of the LTR promoter region. Three functional domains have been identified: an amino terminal amphipathic helix, a cluster of seven cysteine residues, and a stretch of basic amino acids involved in nuclear localization. It is known that conservative mutations of the acidic amino acids of the amphipathic helix are tolerated. Tat mediates the 5xe2x80x2 LTR by interacting with its R region, in a segment termed the xe2x80x9cTARxe2x80x9d (trans-activating response) element (bases 436-497). The xe2x80x9cTARxe2x80x9d element forms a stable stem loop structure that interacts with the Tat protein to prevent premature termination of transcription initiation. Tat is reported in the literature to be absolutely essential for HIV transcription and consequently for viral replication.
The rev gene (CDS 5969..6044, 8369..8643) encodes Rev, another transactivator. Rev is phosphorylated at serine residues, but serine substitution mutants which are not phosphorylated are fully active. The amino terminal 20 amino acids and the carboxy terminal 25 amino acids are known to be dispensable. There are two important domains, a stretch of basic amino acids, which is involved in nuclear localization and in interaction with RRE RNA, and a leucine-rich region, presumed to be involved in transactivation, whose leucines are intolerant of mutation. Rev is a protein whose target is termed RRE (Rev-response element), on the env protein coding region of the mRNA. Interaction of Rev with the RRE region apparently allows for transport of unspliced RNA from the nucleus to the cytoplasm. RRE (7758-7992) is an RNA secondary structure element. Proviruses lacking Rev function remain transcriptionally active but fail to generate new viral particles.
Accessory Genes
The nef gene (CDS 8787..9407) encodes Nef, and overlaps the env gene and the 3xe2x80x2 LTR. Nef may be involved in signal transduction, although this is controversial. There has also been speculation that Nef down-regulates viral expression. The Nef protein does not appear to be essential to the HIV life cycle in tissue culture.
The vif gene (CDS 5041..5619) encodes Vif, the virion infectivity factor. Vif-deficient mutants are typically much less efficient than wild type HIV at cell-free (as opposed to cell-to-cell) virus transmission. It is not a virion component and the mechanism by which it affects infectivity is unclear.
The vpr gene (CDS 5559..5849) encodes Vpr, a virion protein which accelerates the replication and cytopathic effect of HIV-1 in CD4+ T-cells. About 100 copies of Vpr are associated with each virion.
The vpu gene (CDS 6061..6306) encodes Vpu. The vpu gene encodes part of a polycistronic transcript which also includes the env gene. Vpu is a cytoplasmic protein which is thought to facilitate assembly and/or release of viral particles.
PPT (Bases 9059-9075)
Immediately upstream from the 3xe2x80x2 LTR is the polypurine tract vital to initiation of positive-strand DNA synthesis.
3 xe2x80x2LTR (9076..9709)
The 3xe2x80x2 LTR is identical to the 5xe2x80x2 LTR, but is significantly mainly by virtue of its poly-A signal (9602..9607), and the xe2x80x9cRxe2x80x9d repeat sequence (9529..9626) allowing RT jumping during DNA synthesis.
Infectivity
HIV-1 infects activated and resting lymphocytes, terminally differentiated monocytes and neuronal cells through cellular receptors and co-receptors such as CD4, chemokine receptors and galactosyl ceramide (J. M. Harouse et al., Science 253:320-323 [1991]; and R. A. Weiss, Science 272:1885-1886 [1996]). The restricted lentiviral host cell tropism can be expanded by pseudotyping the virus particles with broadly tropic viral envelope proteins from human T cell leukemia virus type I (HTLV-I), amphotropic MLV envelope protein or the vesicular stomatitis virus G glycoprotein (J. C. Burns et al., Proc. Natl. Acad. Sci. USA. 90:8033-8037 [1993]; N. R. Landau et al., J. Virol., 65:162-169 [1991]; K. A. Page et al., J. Virol., 64:5270-5276 [1990]; and D. H. Spector et al., J. Virol., 64:2298-2308 [1990]). Alternatively, a CD4 receptor can be introduced into target cells by adenovirus transduction before HIV vector transduction in a two-step transduction protocol (K. Miyake et al., Human Gene Ther., 7:2281-2286 [1996]). Naldini et al. have demonstrated that HIV-1 vectors pseudotyped with MLV-A or VSV-G envelope could produce up to 5xc3x97105 transducing units/ml of vectors capable of infecting nondividing cells such as macrophages and terminally differentiated neurons (L. Naldini et al., Science 272:263-267. [1996]).
Infection of nondividing cells by lentiviruses such as HIV-1 is mediated by the nuclear localization signal (NLS) in the Gag MA protein (M. I. Bukrinsky et al., Nature 365:666-669 [1993]). Efficient viral entry and integration into non-dividing cells may also require some of the accessory gene products such as Vpr (T. M. Fletcher et al., EMBO J., 15:6155-6165 [1996]; and N. K. Heinzinger et al., Proc. Natl. Acad. Sci. USA. 91:7311-7315 [1994]).
Cytotoxicity
One difficulty related to HIV vector development encountered during the development of the present invention is the cytotoxicity of many HIV gene products to human cells. In particular, it has been difficult to establish continuous cell lines expressing the essential structural proteins Gag, Pol and Env for particle assembly. Cell lines expressing Tat, Rev, Nef have been established. However, expression of Gag, Rev and Vpr has been shown to induce cytopathology, cell death and cell cycle arrest in human cells (See, M. Emerman, Curr. Biol., 6:1096-1103 [1996]; G. Miele and A. M. L. Lever, Gene Ther., 3:357-361 [1995]; and T. Nosaka et al., Exp. Cell. Res,. 209:89-102 [1993]). The development of a tightly inducible system was favored for a lentiviral packaging cell line (H. Yu et al., J. Virol., 70:4530-4537 [1996]). HIV-1 Vpr also induces apoptosis in human cells. The expression of VSV-G protein induces syncytium formation which again is problematic for establishing a packaging cell line.
Other Safety Issues
Unlike other retroviruses, the lentiviruses are able to infect non-dividing cells. Hence, lentiviral vectors have the potential to overcome this limitation of prior vectors systems. However, there is an understandable concern as to the safety of lentiviral vectors, especially those derived from HIV-1. The foremost safety consideration is the risk that either packaging vector and transducing vector will recombine, either with themselves or with defective virus endogenous to the host cell genome, to produce a replication-competent, infectious lentivirus, in particular, replication-competent HIV (RC-HIV). While the vector constructs are replication-defective, the risk of generating RC-HIV is increased with the DNA co-transfection procedure, when a high frequency of recombination events can occur at both DNA and RNA levels. Thus, the packaging constructs and the transducing vectors of lentiviruses could potentially recombine and generate replication-competent viruses (RCV) as do the MLV vectors during co-transfection. However, the chances of generating RCV are reduced if multiple recombination steps are necessary, and if the key envelope gene of HIV-1 is deleted.
Due to the restricted tissue tropism of the native lentiviral env gene, lentiviral vectors were developed that use a pan-tropic envelope gene such as amphotropic MLV env or VSV-Gs. This reduced the possibility of producing a wild-type lentiviral RCV (e.g., an HIV-1 Env-trophic virus). However, it is still possible that an RCV could be generated via recombination with these pan-tropic env genes or endogenous retrotransposon env genes. The fact that human genomes carry numerous human endogenous retroviral sequences (HERVs) further increases the probability of generating a fortuitous recombinant RCV (T. P. Loh et al., J. Virol., 62:4086-4095 [1988]). For example, a recent study demonstrated that a member of the HERV family encodes a protein resembling the lentivirus rev gene product with a nucleolar localization signal, a putative RNA binding domain, and a sequence similar to the Rev effector domain consensus sequence (R. Lower et al., J. Virol., 69:141-149 [1995]).
Some human tissues and cell lines such as the placenta, syncytiotrophoblasts, brain, differentiated U-937 cells, teratocarcinomas, and the mammary carcinoma T47D cells have been shown to express complete human endogenous retrovirus env gene and release retrovirus-like particles. These endogenous retroviruses may form defective particles which lack infectivity. Although the possibility of generating a recombinant RC-HIV with an HERV env gene is low, it is worth examining.
Discussion of Particular Lentiviral Vector Systems
Page, et al., J. Virol., 64: 5270-6 (1990) prepared a noninfectious transducing vector HIV-gpt in which the env gene was replaced with SV-gpt, and a helper vector providing either the HIV-1 gp160 env gene (the HXB2-env vector) or the amphotropic MLV env gene (the SV-A-MLV-env vector).
Shimada, et al., J. Clin. Investig., 88: 1043-7 (1991) describes a recombinant HIV-1 gene transfer system employing two vectors. The packaging vector has a CMV promoter, and an insertion mutation in the packaging signal. The transducing vector replaces part of gag, and all of pol, with a reporter gene cassette. The vector system uses wild type HIV-1 Env proteins to target CD cells. It is worth noting that Shimada et al. state that sequences upstream of gag AUG are important for gag expression, implying that they cannot be modified.
Corbeau, et al, Proc. Nat. Acad. Sci. (USA), 93: 14070-5 (1996) constructed an HIV-1 derived packaging vector by deleting the major packaging signal (37 nucleotides, starting from 6 nt downstream of the 5xe2x80x2 major splice donor site to 7 nucleotides upsteam of the beginning of gag). The genome, which was derived from HIV-1-MN-ST.1 because of its high efficiency of infection in both monocytes and T cells, was otherwise intact. Their transducing vector had the components LTR-gag-RRE-reporter gene (SL3-gpt)-env-LTR. Titers of 10E5 transducing units (TU)/mL were reported.
Corbeau et al. suggest that the first 500 nt of the gag gene may be directly or indirectly involved in the binding of the viral RNA to the nucleocapsid of the virion, and that a stretch within the env gene, including the RRE, also contains a packaging signal.
Corbeau et al. also criticize prior vectors. They attribute the alleged deficiencies of these vectors to the truncation of the vpr gene from the packaging vector, and/or to the deletion of gag and/or env sequences which may contain additional packaging signals from the transducing vector.
Akkina, et al., J. Virol., 70: 2581-5 (1996) demonstrated that an HIV-1 based retroviral vector containing the firefly luciferase reporter gene can be pseudotyped with a broad host range VSV G envelope glycoprotein. The luciferase gene replaced the HIV-1 nef gene. The authors suggested that such a vector should be able to infect CD34+ hematopoietic progenitor cells with high efficiency.
Markowitz, et al, J. Virology 62: 1120-4 (1988) had suggested that viral genes could be separated onto two different plasmids, to provide a safer packaging line for gene transfer Markowitz et al. Placed the gag and pol genes of MLV on one plasmid, and the env gene on the other. The plasmids had deletions of the 3xe2x80x2LTRs and the packaging signal as well. Hence, to generate intact retrovirus, there would need to be several recombination events. Markowitz"" strategy was adapted to HIV-1 by Naldini et al., as described below.
Naldini, et al., Science, 272: 263 (1996) describes a lentiviral vector-based system for gene delivery. There are three vectors in the system. The first packaging vector (pCMVxcex94R9) provides the HIV gag, pro, pol, vif, nef, tat, rev, and vpr genes, but the env and vpu genes, and the packaging signal, were inactivated. (A later paper, cited below, makes it clear that the env gene was inactivated by insertion of a linker containing multiple stop codons.) The human cytomegalovirus (CMV) immediate early promoter was substituted for the 5xe2x80x2 LTR, while the 3xe2x80x2 LTR was replaced with a polyA site from the human insulin gene. The major splice donor site was preserved. A second packaging vector was used to broaden the tropism of the vector system. In one variant, this vector expressed the amphotropic envelope of Moloney leukemia virus (MLV), under control of the MLV LTR, and in the other, it expressed the G glycoprotein of vesicular stomatitis virus (VSV) under the direction of the CMV promoter. (The lternative Env protein was the only expression product of the second vector.) The final element of the system was a transducing vector (pHRxe2x80x2), providing, in order, the 5xe2x80x2LTR, the major splice donor site, the major packaging signal, nearly 350 base pairs of gag, the env sequence encompassing the RRE element, a splice acceptor site, an internal CMV promoter, a reporter gene (luciferase or beta-galactosidase), and a 3xe2x80x2 LTR.
Naldini et al., Proc. Nat. Acad. Sci. (USA), 93: 11382-8 (1996) discuss the use of VSV-G-pseudotyped lentiviral vector particles to achieve xe2x80x9clong-termxe2x80x9d expression of a transgene in adult rat brains injected with the particles. The packaging vector differs from that described above in that 1.4 kbp was deleted from the env gene, downstream of the functional vpu gene, and replaced with an inframe stop codon. See also Blomer et al., J. Virol., 71: 6641-9 (September 1997).
Sodroski, U.S. Pat. No. 5,654,195 (1997) describes a hybrid virus in which the 5xe2x80x2 DNA segment encodes functional SIV or HIV-2 gag, pol, pro, vif, and vpx proteins, and the 3xe2x80x2 DNA segment encodes functional HIV-1 env, tat and rev proteins, and a functional SIV or HIV-2 nef protein. The 5xe2x80x2 and 3xe2x80x2 LTRs are from SIV or HIV-2.
Sodroski, U.S. Pat. No. 5,665,577 (1997) discloses an HIV vector which comprises the gag, pol and env genes but lacks the HIV major packaging, signal identified therein as AAAAATTTTGACTAGCGGA(SEQ ID NO: 4). When introduced into a eukaryotic host cell, these express the structural proteins to form HIV virions that do not contain sufficient HIV RNA to result in a replication-competent HIV virion.
The present invention contemplates attenuated lentiviruses, and improved viral packaging and transducing vectors derived from lentiviruses, especially HIV-1, and useful for the delivery of nonlentiviral genes to target cells. It also contemplates the use of these vectors in delivering transgenes to target cells, especially nondividing cells, in organisms, especially humans.
Packaging Vectors
The packaging vectors of the present invention differ from those known previously in that they contain less in the way of lentiviral sequences from a single lentivirus, and hence present a reduced risk of recombination. In particular, the packaging vectors of the present invention are characterized by either the use of a modified but functional major splice donor site, substantially incapable of serving as a site for homologous recombination, or by the complete omission of the major splice donor site. In a preferred embodiment, the modified major splice donor site is modified so that it is substantially identical to the major splice donor site of a non-lentiviral retrovirus, especially that of Rous Sarcoma Virus (RSV).
Preferably, other non-essential sequences, such as the accessory genes, of the source lentivirus are also deleted in the course of the construction of the packaging vector. Preferably, in the 5xe2x80x2 LTR region of the packaging vector, the wild-type promoter and enhancer are replaced with a nonhomologous promoter (and, optionally, a nonhomologous enhancer). These changes likewise serve to reduce the risk of generating replication-competent virus through recombination with the transducing vector or a defective provirus endogenous to the host or target cell.
Preferably, the 5xe2x80x2 LTR promoter is an tightly inducible promoter, so that expression of Gag, Pol and Env proteins is under the control of the biologist. This, together with the inactivation of certain accessory genes, tends to reduce cytotoxicity.
Preferably, the Gag and Pol functions are encoded by one vector and the Env functions (preferably, a non HIV-1-like envelope protein) by another vector.
Preferably, gag expression is enhanced by the operable linking of the gag gene to a Kozak sequence.
Transducing Vectors
In a preferred embodiment, the transducing vector likewise is characterized by a functional major splice donor site which differs from that of its source lentivirus. In the latter case, its major splice donor site need not be identical to that of the packaging vector(s). The modification should leave a functional packaging signal, too.
Preferably, it likewise has a strong nonlentiviral promoter/enhancer in place of the normal 5xe2x80x2 LTR.
Preferably, the gag (except for packaging signals) and pol sequences are deleted. Desirably, the env sequences are deleted to the extent that this can be done without a substantial loss in yield.
While there may still be regions of sequence identity between the packaging and transducing vectors which are sufficiently long to present a meaningul risk of homologous recombination, a characteristic of the preferred vector system is that homologous recombination alone, among only the packaging and transducing vectors, cannot create a recombinant virus which possesses, simultaneously, a functional packaging signal, a functional major splice donor site, and a gag AUG, even if the recombined virus possesses a 5xe2x80x2 promoter/enhancer and genes otherwise encoding equivalents of the Gag, Pol and Env proteins. The first region of significant homology is in the gag gene, after the initiation codon. Hence, if the recombinant virus derives a functional packaging signal and a functional major splice donor site from the transducing vector, it will lack the gag AUG, since it can crossover to the packaging vector only after the AUG. Contrariwise, if it has the 5xe2x80x2 sequence of the packaging vector through the gag AUG, it will lack a functional packaging signal and a functional major splice donor site. Of course, a replication-competent virus could still be generated by nonhomologous recombination, or by further recombination with a defective endogenous retrovirus.
Certain speculative vector systems are also described herein which further increase safety.